(a) Field of the Invention
The present invention relates to a calcium- and phospholipid-dependent protein phosphorylation enzyme (hereinafter referred to as protein phosphorylation enzyme C or protein kinase C for brevity) and to recombinant DNA technique for the production thereof, especially to rat kinase C and human kinase C.
(b) Description of the Prior Art
Numerous kinds of biologically active substances such as cell growth factors, hormones and neutrotransmitters are known to participate in the exertion of various functions and adaptation phenomena of living organisms. These extracellular signals are exerted through intracellular mediators such as cyclic AMP (cAMP), cyclic GMP (cGMP), diacylglycerol (DG) and calcium (Ca.sup.++). It has been revealed that DG is produced as a result of inositol phospholipid breakdown in cell membranes by a number of hormones and neutrotransmitters which are responsible for the activation of cellular functions. It is considered that the activation of various cellular functions and cellular proliferation are attained by the phosphorylation of various intracellular proteins with a protein kinase C enzyme which is activated by DG in the presence of Ca.sup.++. Thus, protein kinase C is an enzyme which plays an important role in a mechanism of main information transduction of external signals. The enzyme isolated from rat brains is an acidic protein having an isoelectric point of pH 5.6 and a molecular weight of approximate 77,000. This enzyme is composed of a single peptide having a hydrophobic region serving as a binding site to cell membranes and a hydrophilic region in which the active center exists. The protein kinase C is normally in an inactive form but is activated by calcium, phosphatidyl-serine and DG. ATP is known to serve as the phosphoric acid donor.
Thus, the protein kinase C performs the transduction of extracellular signals into cells through the phosphorylation of proteins as a protein kinase and, therefore, it is one of the indispensable enzymes in studying of extracellular signal reception and transduction mechanism and is a very valuable reagent. The enzyme is also activated directly by tumor-promoting phorbol esters. Phorbol esters are known not only to cause promotion of carcinogenesis but also to take part in various biological reactions such as mitosis and differentiation of cells, induction of enzymes and acceleration of lipid metabolism. In these circumstances, there is a great possibility that the protein kinase C becomes an important index enzyme in diseased cells or tumor cells resulting from troubles in cellular signal transduction mechanism and that antibodies to the protein kinase C are used as diagnostic and inspection reagents. For these purposes, it is essential to provide a large amount of human protein kinase C. However, natural human protein kinase C is present only in a small amount. Further, to obtain the human protein kinase C from human tissues involves various problems and is extremely difficult. Therefore, the amino acid sequence of the human protein kinase C and the DNA sequence of the human protein kinase C gene have not been determined yet.
On the other hand, protein kinase C may be derived from readily available animal parts, such as, for example, a protein kinase C purified from rat brains. However, the amino acid sequence and the gene DNA sequence of rat protein kinase C has not been determined yet. It is also very difficult to obtain rat protein kinase C in a large amount.
As described above, there are remaining a number of unknown points with respect to the properties, the amino acid sequence and the gene of human protein kinase C and rat protein kinase C. Therefore, it is highly desired to elucidate the gene coding for the protein kinase C and to provide a production method for the protein as a reagent and an inspecting agent by gene recombinant techniques.